Disease resistant plants

ABSTRACT

The present invention relates to a plant, which is resistant to a pathogen of viral, bacterial, fungal or oomycete origin, wherein the plant has a reduced level, reduced activity or complete absence of DMR6 protein as compared to a plant that is not resistant to the said pathogen, in particular organisms of the Fungi or the phylum Oomycota. The invention further relates to a method for obtaining a plant, which is resistant to a pathogen of viral, bacterial, fungal or oomycete origin, comprising reducing the endogenous level or activity of DMR6 protein in the plant. In addition, the invention relates to the use of a DMR6 promotor for providing disease resistant plants.

This application is a divisional application of co-pending U.S. patentapplication Ser. No. 14/528,707, filed Oct. 30, 2014, which is adivisional application of U.S. patent application Ser. No. 14/250,875,filed Apr. 11, 2014 and issued as U.S. Pat. No. 9,121,029, which is adivisional application of U.S. patent application Ser. No. 12/525,236,filed Dec. 22, 2009 and issued as U.S. Pat. No. 8,742,207, which is theU.S. national phase of PCT Application No. PCT/EP2008/000718, filed Jan.30, 2008, which claims priority to PCT Application No.PCT/EP2007/050976, filed Feb. 1, 2007, each of which is incorporatedherein by reference in their entirety.

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety. The name of the text filecontaining the Sequence Listing is 1601595 ST25.txt. The size of thetext file is 108,966 bytes, and the text file was created on May 12,2016.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to disease resistant plants, in particularplants resistant to organisms of the kingdom Fungi and the phylumOomycota, the oomycetes. The invention further relates to plant genesconferring disease resistance and methods of obtaining such diseaseresistant plants for providing protection to Oomycota pathogens.

Description of Related Art

Resistance of plants to fungal and oomycete pathogens has beenextensively studied, for both pathogen specific and broad resistance. Inmany cases resistance is specified by dominant genes for resistance.Many of these race-specific or gene-for-gene resistance genes have beenidentified that mediate pathogen recognition by directly or indirectlyinteracting with avirulence gene products or other molecules from thepathogen. This recognition leads to the activation of a wide range ofplant defense responses that arrest pathogen growth.

In plant breeding there is a constant struggle to identify new sourcesof mostly monogenic dominant resistance genes. In cultivars with newlyintroduced single resistance genes, protection from disease is oftenrapidly broken, because pathogens evolve and adapt at a high frequencyand regain the ability to successfully infect the host plant. Therefore,the availability of new sources of disease resistance is highly needed.

Alternative resistance mechanisms act for example through the modulationof the defense response in plants, such as the resistance mediated bythe recessive mlo gene in barley to the powdery mildew pathogen Blumeriagraminis fsp. hordei. Plants carrying mutated alleles of the wildtypeMLO gene exhibit almost complete resistance coinciding with the abortionof attempted fungal penetration of the cell wall of single attackedepidermal cells. The wild type MLO gene thus acts as a negativeregulator of the pathogen response. This is described in WO9804586.

Other examples are the recessive powdery mildew resistance genes, foundin a screen for loss of susceptibility to Erysiphe cichoracearum. Threegenes have been cloned so far, named PMR6, which encodes a pectatelyase-like protein, PMR4 which encodes a callose synthase, and PMR5which encodes a protein of unknown function. Both mlo and pmr genesappear to specifically confer resistance to powdery mildew and not tooomycetes such as downy mildews.

Broad pathogen resistance, or systemic forms of resistance such as SAR,has been obtained by two main ways. The first is by mutation of negativeregulators of plant defense and cell death, such as in the cpr, lsd andacd mutants of Arabidopsis. The second is by transgenic overexpressionof inducers or regulators of plant defence, such as in NPR1overexpressing plants.

The disadvantage of these known resistance mechanisms is that, besidespathogen resistance, these plants often show detectable additional andundesirable phenotypes, such as stunted growth or the spontaneousformation of cell death.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a form of resistancethat is broad, durable and not associated with undesirable phenotypes.

In the research that led to the present invention, an Arabidopsisthaliana mutant screen was performed for reduced susceptibility to thedowny mildew pathogen Hyaloperonospora parasitica. EMS-mutants weregenerated in the highly susceptible Arabidopsis line Ler eds1-2. Eightdowny mildew resistant (dmr) mutants were analysed in detail,corresponding to 6 different loci. Microscopic analysis showed that inall mutants H. parasitica growth was severely reduced. Resistance ofdmr3, dmr4 and dmr5 was associated with constitutive activation of plantdefense. Furthermore, the dmr3 and dmr4, but not dmr5 mutants, were alsoresistant to Pseudomonas syringae and Golovinomyces orontii.

In contrast, enhanced activation of plant defense was not observed inthe dmr1, dmr2, and dmr6 mutants. The results of this research have beendescribed in Van Damme et al. (2005) Molecular Plant-MicrobeInteractions 18(6) 583-592. This article does not disclose theidentification and characterization of the DMR genes.

The dmr6 mutant was identified in a loss-of-susceptibility screen in theArabidopsis Ler eds1-2 background. The DMR6 gene now has been cloned andcharacterized. Thus, it was found that DMR6 is the gene At5g24530,encoding for an oxidoreductase (DNA and amino acid sequence are depictedin FIG. 2). Oxidoreductases are enzymes that catalyze the transfer ofelectrons from one molecule, the oxidant, to another, the reductant.According to the present invention, it has been found that lack of afunctional DMR6 protein results in downy mildew resistance.

The present invention thus provides a plant, which is resistant to apathogen of viral, bacterial, fungal or oomycete origin, characterizedin that the plant has a reduced level, reduced activity or completeabsence of the DMR6 protein as compared to a plant that is not resistantto the said pathogen.

This form of resistance is in particular effective against pathogens ofthe phylum Oomycota, such as Albugo, Aphanomyces, Basidiophora, Bremia,Hyaloperonospora, Pachymetra, Paraperonospora, Perofascia,Peronophythora, Peronospora, Peronosclerospora, Phytium, Phytophthora,Plasmopara, Protobremia, Pseudoperonospora, Sclerospora, Viennotiaspecies, as well as to pathogens belonging to the Fungi.

The resistance according to the invention is based on an altered, inparticular a reduced level, reduced activity or complete absence of theDMR6 protein in planta. The term “DMR6 protein” in this respect relatesto the DMR6 gene product, such as the protein encoded by the At5g24530gene in Arabidopsis. Such alterations can be achieved in various ways.

In one embodiment of the invention, the reduced level of DMR6 protein isthe result of a reduced endogenous DMR6 gene expression. Reducing theexpression of the DMR6 gene can be achieved, either directly, such as bygene silencing, or indirectly by modifying the regulatory sequencesthereof, or by stimulating repression of the gene.

Modulating the DMR6 gene to lower its activity or expression can beachieved at various levels. First, the endogenous gene can be directlymutated. This can be achieved by means of a mutagenic treatment.Alternatively, a modified DMR6 gene can be brought into the plant bymeans of transgenic techniques or by introgression, or the expression ofDMR6 can be reduced at the regulatory level, for example by modifyingthe regulatory sequences or by gene silencing.

In another embodiment of the invention, the reduced level of DMR6protein is the result of a mutation in the DMR6 gene resulting in areduced DMR6 expression as compared to the wild-type DMR6 gene whereinno such mutation is present, or resulting in a reduced mRNA or proteinstability. In a particular embodiment this is achieved by mutations inthe DMR6 coding sequence that result in a non-functional DMR6 protein,i.e. without or with reduced enzymatic activity.

In another embodiment of the invention, reduced expression can beachieved by down-regulation of DMR6 gene expression either at thetranscriptional or the translational level, e.g. by gene silencing or bymutations that affect the expression of the DMR6 gene.

This invention is based on research performed on resistance toHyaloperonospora parasitica in Arabidopsis but is a general concept thatcan be more generally applied in plants, in particular in crop plantsthat are susceptible to infections with pathogens, such as Oomycota andFungi.

The invention is suitable for a large number of plant diseases caused byoomycetes such as, but not limited to, Bremia lactucae on lettuce,Peronospora farinosa on spinach, Pseudoperonospora cubensis on membersof the Cucurbitaceae family, e.g. cucumber and melon, Peronosporadestructor on onion, Hyaloperonospora parasitica on members of theBrasicaceae family, e.g. cabbage, Plasmopara viticola on grape,Phytophthora infestans on tomato and potato, and Phytophthora sojae onsoybean.

When the modification of DMR6 gene expression in a plant is to beachieved via genetic modification of the DMR6 gene or via theidentification of mutations in the DMR6 gene, and the gene is not yetknown it must first be identified. To generate pathogen-resistantplants, in particular crop plants, via genetic modification of the DMR6gene or via the identification of mutations in the DMR6 gene, theorthologous DMR6 genes must be isolated from these plant species.

Various methods are available for the identification of orthologoussequences in other plants.

A method for the identification of DMR6 orthologous sequences in a plantspecies, may for example comprise identification of DMR6 ESTs of theplant species in a database; designing primers for amplification of thecomplete DMR6 transcript or cDNA; performing amplification experimentswith the primers to obtain the corresponding complete transcript orcDNA; and determining the nucleotide sequence of the transcript or cDNA.Suitable methods for amplifying the complete transcript or cDNA insituations where only part of the coding sequence is known are theadvanced PCR techniques 5′RACE, 3′RACE, TAIL-PCR, RLM-RACE andvectorette PCR.

Alternatively, if no nucleotide sequences are available for the plantspecies of interest, primers are designed on the DMR6 gene of a plantspecies closely related to the plant of interest, based on conserveddomains as determined by multiple nucleotide sequence alignment, andused to PCR amplify the orthologous sequence. Such primers are suitablydegenerate primers.

Another reliable method to assess a given sequence as being a DMR6ortholog is by identification of the reciprocal best hit. A candidateorthologous DMR6 sequence of a given plant species is identified as thebest hit from DNA databases when searching with the Arabidopsis DMR6protein or DNA sequence, or that of another plant species, using a Blastprogram. The obtained candidate orthologous nucleotide sequence of thegiven plant species is used to search for homology to all Arabidopsisproteins present in the DNA databases (e.g. at NCBI or TAIR) using theBlastX search method. If the best hit and score is to the ArabidopsisDMR6 protein, the given DNA sequence can be described as being anortholog, or orthologous sequence.

DMR6 is encoded by a single gene in Arabidopsis as deduced from thecomplete genome sequence that is publicly available. In the genome ofrice 3 orthologs, and in poplar 2 orthologs have been identified. Inmost other plant species tested so far, DMR6 appears to be encoded by asingle gene, as determined by the analysis of mRNA sequences and ESTdata from public DNA databases. The orthologous genes and proteins areidentified in these plants by nucleotide and amino acid comparisons withthe information that is present in public databases.

Alternatively, if no DNA sequences are available for the desired plantspecies, orthologous sequences are isolated by heterologoushybridization using DNA probes of the DMR6 gene of Arabidopsis oranother plant or by PCR methods, making use of conserved domains in theDMR6 coding sequence to define the primers. For many crop species,partial DMR6 mRNA sequences are available that can be used to designprimers to subsequently PCR amplify the complete mRNA or genomicsequences for DNA sequence analysis.

In a specific embodiment the ortholog is a gene of which the encodedprotein shows at least 50% identity with the Arabidopsis DMR6 protein(At5g24530) or that of other plant DMR6 proteins. In a more specificembodiment the identity is at least 55%, more specifically 60%, evenmore specifically 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D shows the alignment of the amino acid sequences of the DMR6protein of Arabidopsis thaliana (SEQ ID NO. 62) and orthologs fromAquilegia species (SEQ ID NO. 63), Citrus sinensis (SEQ ID NO. 64),Coffea canephora (SEQ ID NO. 65), Cucumis sativus (SEQ ID NO. 67),Gossypium hirsitum (SEQ ID NO. 68), Lactuca sativa (SEQ ID NO. 70),Medicago truncatula (SEQ ID NO. 71), Oryza sativa (SEQ ID NOs. 72-74),Populus trichocarpa (SEQ ID NOs. 75 and 76), Solanum lycopersicum (SEQID NOs. 77 and 78), Sorghum bicolor (SEQ ID NO. 79), Spinacia oleracea(SEQ ID NO. 81), Vitis vinifera (SEQ ID NO. 82), Zea mays (SEQ ID NO.83), and Zingiber officinale (SEQ ID NO. 84), using the CLUSTAL W (1.83)multiple sequence alignment programme (EBI). Below the sequences theconserved amino acids are indicated by the dots, and the identical aminoacids are indicated by the asterisk.

FIG. 2 shows the nucleotide (SEQ ID NO. 61) and amino acid sequence (SEQID NO. 62) of the DMR6 gene (At5g24530, gi 42568064, Genbank NM 122361)and protein (gi 15238567, Genbank NP 197841) of Arabidopsis thaliana,respectively.

FIG. 3 shows the nucleotide (SEQ ID NO. 69) and derived amino acidsequence (SEQ ID NO. 70) of the DMR6 ortholog of Lactuca sativa,respectively.

FIG. 4 shows the nucleotide (SEQ ID NO. 80) and derived amino acidsequence (SEQ ID NO. 81) of the DMR6 ortholog of Spinacia oleracea,respectively.

FIG. 5 shows the nucleotide (SEQ ID NO. 66) and derived amino acidsequence (SEQ ID NO. 67) of the DMR6 ortholog of Cucumis sativus andCucumis melo.

FIG. 6A-B shows the downy mildew resistance of the Arabidopsis dmr6mutants. (a) Quantification of sporangiophores of H. parasitica isolateWaco9, 7 days post inoculation, on the dmr6-1 mutant (BC₂, line E37)compared to its parental line Ler eds1-2 and on the dmr6-2 mutant (FLAG445D09 T-DNA line) compared to its parental line Ws-4. (b) Restorationof susceptibility by complementation with the At5g24530 gene in thedmr6-1 mutant. H. parasitica spores per mg seedling weight werequantified on Ler eds1-2, dmr6-1 and 5 complementation lines (#121, 122,211,231, and 241).

FIG. 7 shows the structure of the Arabidopsis DMR6 gene and dmr6-1 anddmr6-2 mutations. The DMR6 gene contains four exons and a codingsequence of 1026 bases. The two alleles are indicated; dmr6-1 with abase change in exon 2, and dmr6-2 with a T-DNA insertion into intron 2.

FIG. 8 shows the relative transcript levels of DMR6 in Ler plants eithermock treated or inoculated with a compatible or incompatible H.parasitica isolate. Transcript levels were determined at different dayspost inoculation. The difference in cycle threshold (ΔCT) values reflectthe number of additional PCR amplification cycles required to reach anarbitrary threshold product concentration as compared to ΔCTIN2. A lowerΔCT value indicates a higher transcript level.

FIG. 9 shows the expression of the DMR6 promoter-reporter (pDMR6::GUS)construct in transgenic Arabidopsis lines, visualized with only X-glucas substrate (FIG. d and e) or Magenta-Xgluc (FIG. a-c) and trypan bluestaining of H. parasitica growth (a) Ler eds1-2 (pDMR6::GUS) 3 dpi withH. parasitica, Cala2 isolate. (b) Col-0 (pDMR6::GUS) 3 dpi with H.parasitica, Waco9 isolate. (c) Ler eds1-2 (pDMR6::GUS) 3 dpi with H.parasitica, Emoy2 isolate. (d) Col-0 (pDMR6::GUS) 3 dp wounding. (e)Col-0 (pDMR6::GUS) 3 dp BTH application.

FIG. 10A-B shows the Q-PCR analysis of the transcript levels of thegenes; At4g14365, At1g14880, ACD6, PR-1, PR-2 and PR-5, selected as upregulated in the dmr 6-1 micro array analysis. (a) Transcription levelsof the six genes in dmr6-1 compared to Ler eds1-2 and additionally theDMR6 transcript. (b) Elevated gene transcripts of six defence-associatedgenes in dmr6-2 versus Ws-4. ΔCT reflects the number of additional PCRamplification cycles required to reach the level of ΔCTIN2 transcripts.A lower ΔCT value indicates a higher transcript level.

FIG. 11 shows the nucleotide sequence (SEQ ID NO. 107) of the 3 kbregion upstream of the start codon of the DMR6 gene (at5g24530) ofArabidopsis thaliana, including the promoter and 5′-UTR (underlined).

FIG. 12 shows the nucleotide (SEQ ID NO. 95) and derived amino acidsequence (SEQ ID NO. 96) of the DMR6 ortholog of Solanum lycopersicum,respectively.

FIG. 13 shows the nucleotide (SEQ ID NO. 97) and derived amino acidsequence (SEQ ID NO. 98) of the DMR6 ortholog of Nicotiana benthamiana,respectively.

FIG. 14 shows complementation of Arabidopsis thaliana dmr6-1 with DMR6derived from Cucumis sativa (Cs), Spinacia oleracea (Si), Lactuca sativa(Ls) and Solanum lycopersicum (So).

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows orthologous DMR6 sequences (described in Table 1) that havebeen identified in publicly available databases and obtained by PCRamplification on cDNA and subsequent sequencing. After orthologous DMR6sequences are identified, the complete nucleotide sequence of theregulatory and coding sequence of the gene is identified by standardmolecular biological techniques. For this, genomic libraries of theplant species are screened by DNA hybridization or PCR with probes orprimers derived from a known DMR6 gene to identify the genomic clonescontaining the DMR6 gene. Alternatively, advanced PCR methods, such asRNA ligase-mediated RACE (RLM-RACE), can be used to directly amplifygene and cDNA sequences from genomic DNA or reverse-transcribed mRNA.DNA sequencing subsequently results in the characterization of thecomplete gene or coding sequence.

Once the DNA sequence of the gene is known this information is used toprepare the means to modulate the expression of the DMR6 gene.

To achieve a reduced DMR6 protein level, the expression of the DMR6 genecan be down-regulated or the enzymatic activity of the DMR6 protein canbe reduced by amino acid substitutions resulting from nucleotide changesin the DMR6 coding sequence.

In a particular embodiment of the invention, downregulation of DMR6 geneexpression is achieved by gene-silencing using RNAi. For this,transgenic plants are generated expressing a DMR6 anti-sense construct,an optimized micro-RNA construct, an inverted repeat construct, or acombined sense-anti-sense construct, so as to generate dsRNAcorresponding to DMR6 that leads to gene silencing.

In an alternative embodiment, one or more regulators of the DMR6 geneare downregulated (in case of transcriptional activators) by RNAi.

In another embodiment regulators are upregulated (in case of repressorproteins) by transgenic overexpression. Overexpression is achieved in aparticular embodiment by expressing repressor proteins of the DMR6 genefrom a strong promoter, e.g. the 35S promoter that is commonly used inplant biotechnology.

The downregulation of the DMR6 gene can also be achieved by mutagenesisof the regulatory elements in the promoter, terminator region, orpotential introns. Mutations in the DMR6 coding sequence in many casesleads to amino acid substitutions or premature stop codons thatnegatively affect the expression or activity of the encoded DMR6protein.

These mutations are induced in plants by using mutagenic chemicals suchas ethyl methane sulfonate (EMS), by irradiation of plant material withgamma rays or fast neutrons, or by other means. The resulting nucleotidechanges are random, but in a large collection of mutagenized plants themutations in the DMR6 gene can be readily identified by using theTILLING (Targeting Induced Local Lesions IN Genomes) method (McCallum etal. (2000) Targeted screening for induced mutations. Nat. Biotechnol.18, 455-457, and Henikoff et al. (2004) TILLING. Traditional mutagenesismeets functional genomics. Plant Physiol. 135, 630-636). The principleof this method is based on the PCR amplification of the gene of interestfrom genomic DNA of a large collection of mutagenized plants in the M2generation. By DNA sequencing or by looking for point mutations using asingle-strand specific nuclease, such as the CEL-I nuclease (Till et al.(2004) Mismatch cleavage by single-strand specific nucleases. NucleicAcids Res. 32, 2632-2641) the individual plants that have a mutation inthe gene of interest are identified.

By screening many plants, a large collection of mutant alleles isobtained, each giving a different effect on gene expression or enzymeactivity. The gene expression or protein levels can for example betested by analysis of DMR6 transcript levels (e.g. by RT-PCR) or byquantification of DMR6 protein levels with antibodies.

Plants with the desired reduced DMR6 level or DMR6 expression are thenback-crossed or crossed to other breeding lines to transfer only thedesired new allele into the background of the crop wanted.

The invention further relates to mutated DMR6 genes. In a particularembodiment, the invention relates to dmr6 alleles with premature stopcodons, such as the dmr6-1 allele.

In another embodiment, the invention relates to mutated versions of theDMR6 genes of Lactuca sativa, Cucumis sativus, and Spinacia oleracea asshown in FIGS. 3-5.

The present invention demonstrates that plants having no or a reducedlevel of functional DMR6 gene product show resistance to pathogens, inparticular of oomycete and fungal origin. With such knowledge theskilled person can identify so far unknown natural variants of a givenplant species that have variants of the DMR6 gene that lead to a reducedlevel or absence of a functional DMR6 protein, or mutated versions ofthe DMR6 protein, and to use these natural variants according to theinvention.

The present invention further relates to the use of a DMR6 promotor forproviding disease resistance into plants, i.e. for providing plants witha resistance to a pathogen of viral, bacterial, fungal or oomyceteorigin. According to the present invention, the transcriptionalup-regulation of DMR6 in response to pathogen infection has beendemonstrated. Both transcript analysis as well as promotor DMR6-reporterlines support this finding (see Example 1, below). Thepathogen-inducible DMR6 promotor according to the invention thus isparticularly useful to control the expression of inducible systems thatlead to disease resistance in plants.

One example of such inducible system that leads to disease resistance inplants, and in which the DMR6 promotor according to the presentinvention may be effective, has e.g. been described in WO 99/45125,wherein an antisense nucleotide sequence for a gene involved in theregulation of the C-5 porphyrin metabolic pathway is operably linked toa pathogen-inducible promotor and used to transform plant cells.Expression of the antisense nucleotide sequence in response to thepathogen effectively disrupts porphyrin metabolism of the transformedplant cell, and development of a localized lesion wherein the spread ofthe pathogen is contained. WO 96/36697 also discloses inducible systemsleading to disease resistance in plants, wherein an inducible promotorcontrols the expression of a protein capable of evoking thehypersensitivity response in a plant. EP 0474857 furthermore discloses amethod for the induction of pathogen resistance in plants, comprisingtransforming plants with polynucleotide sequences encoding a pair ofpathogen-derived-avirulence-gene/plant-derived-resistance gene, whereinthe expression of one of or both the elicitor peptide and the resistancegene is regulated by a pathogen inducible promotor. Further examples ofinducible systems leading to resistance to pathogens in plants have beendescribed in e.g. WO 98/32325.

In a particular preferred embodiment, the present invention relates to amethod of providing disease resistance in a plant, comprisingtransforming a plant cell with a DNA construct comprising at least oneexpressible nucleic acid which is operably linked to apathogen-inducible promotor that is operable within a plant cell, andregenerating transformed plants from said plant cells, wherein thepathogen-inducible promotor is a DMR6 promotor, and wherein theexpression of the expressible nucleic acid confers disease resistance tothe transgenic plant.

The invention also relates to disease resistance plants, obtainable bysaid method, as well as to plant tissue, and seeds obtained from saidplants.

The invention in particular relates to plants, which are resistant to apathogen of viral, bacterial, fungal or oomycete origin, wherein theplant comprises in its genome a DNA construct, comprising at least oneexpressible nucleic acid which is operably linked to apathogen-inducible promotor, wherein the pathogen-inducible promotor isa DMR6 promotor.

The present invention also relates to the DNA construct per se,comprising at least one expressible nucleic acid which is operablylinked to a pathogen-inducible promotor, wherein the pathogen-induciblepromotor is a DMR6 promotor. The construct of the invention can be usedto transform plant cells which may be regenerated into transformedplants. Furthermore, transformed plant tissue and seed may be obtained.Suitable methods for introducing the construct of the invention intoplant cells are known to the skilled person.

According to the invention, by “operably linked” is meant that apromotor and an expressible nucleic acid, e.g. a gene, are connected insuch way as to permit initiation of transcription of the expressiblenucleic acid (e.g. gene) by the promotor.

By “expressible nucleic acid” is meant a nucleic acid (e.g. a gene, orpart of a gene) that can be expressed in the cell, i.e. that can betranscribed into mRNA, and eventually may be translated into a protein.The expressible nucleic acid may be genomic DNA, cDNA, or chemicallysynthesized DNA or any combination thereof.

According to the present invention, a DNA construct comprises allnecessary nucleic acid elements which permit expression (i.e.transcription) of a particular nucleic acid in a cell. Typically, theconstruct includes an expressible nucleic acid, i.e. a nucleic acid tobe transcribed, and a promotor. The construct can suitably beincorporated into e.g a plasmid or vector.

The expressible nucleic acid preferably is a gene involved in a plantdefence response, e.g. a gene associated with the hypersensitivityresponse of a plant. In the hypersensitivity response (HR) of a plant,the site in the plant where the pathogen invades undergoes localizedcell death by the induced expression of a suicide mechanism thattriggers said localized cell death in response to pathogens. In thisway, only a few plant cells are sacrificed and the spread of thepathogen is effectively arrested. Examples of said genes involved in aplant defence response are the regulatory protein NPR1/NIM1 (Friedrichet al., Mol. Plant Microbe Interact. 14(9): 1114-1124, 2001) and thetranscription factor MYB30 (Vailleau et al., Proc. Natl. Acad. Sci. USA99(15): 10179-10184, 2002).

In a particular embodiment, the expressible nucleic acid encodes anautologous or heterologous polypeptide capable of conferringdisease-resistance to a plant. By “autologous polypeptide” is meant anypolypeptide that is expressed in a transformed plant cell from a genethat naturally occurs in the transformed plant cell. By “heterologouspolypeptide” is meant any polypeptide that is expressed in a transformedplant cell from a gene that is partly or entirely foreign (i.e. does notnaturally occur in) to the transformed plant cell. Examples of suchpolypeptides are the mammalian Bax protein, which encodes apro-apoptotic protein and results in cell death in plants (Lacomme andSanta Cruz, Proc. Natl. Acad. Sci. USA 96(14): 7956-61, 1999) and fungalchitinases (de las Mercedes Dana et al., Plant Physiol. 142(2): 722-730,2006).

Preferably, the DMR6 promotor is the Arabidopsis DMR6 promotor. The DMR6promotor comprises a region of 3000 bp that is upstream of theArabidopsis DMR6 coding sequence (ATG start codon) and includes the5′UTR. Preferably the DMR6 promotor comprises a nucleotide sequence asdefined in FIG. 11, and/or any functional fragment thereof, i.e. anyfragment (or part) of said sequence which still is capable of initiatingtranscription of the expressible nucleic acid(s) to which it is operablylinked, and/or natural variants thereof, i.e. natural variants of thispromotor which may contain small polymorphisms, but which are generallyat least 90% identical.

In a further preferred embodiment, the DMR6 promotor is an orthologousDMR6 promotor, i.e. a promotor of an orthologous DMR6 gene. Methods foridentifying DMR6 orthologs have been described in Example 2 below. Oncethe DMR6 orthologs have been identified, the skilled person will be ableto isolate the respective promotor of said orthologs, using standardmolecular biological techniques.

According to the present invention, the DMR6 promotor has been shown tobe strongly pathogen-induced, and the DMR6 promotor is not highlyexpressed in other non-infected tissues. Thus, it is a very suitablepromotor for use in inducible systems for providing resistance topathogens of viral, bacterial, fungal or oomycete origin in plants.Examples of specific pathogens and plants for which the induciblesystem, using the DMR6 promotor of the present invention, suitably canbe used, have been given above.

The present invention is illustrated in the following examples that arenot intended to limit the invention in any way. In the examplesreference is made to the figures described above and the followingtables.

Table 1 shows the Genbank accession numbers and GenInfo identifiers ofthe Arabidopsis DMR6 mRNA and orthologous sequences from other plantspecies.

Table 2 shows the PCR primers for the markers used for the map-basedcloning of DMR6.

Table 3 shows primer pairs for cloning dmr6 orthologs in a suitableplant expression vector.

Example 1 The Arabidopsis DMR6 (At5g24530) Gene is Required for DownyMildew Susceptibility Experimental Procedures

Hyaloperonospora parasitica Growth and Infection

H. parasitica isolate Waco9 was provided by Dr. M. Aarts (WUR,Wageningen, N L) and isolate Cala2 provided by Dr. E. Holub (Warwick H RI, Wellsbourne, UK) and maintained on Arabidopsis Ws-0 and Ler,respectively. Inocula (400,000 spores per ml) were weekly transferred to10 day old healthy seedlings (Holub, E. B. et al., Mol. Plant MicrobeInteract. 7: 223-239, 1994) by use of a spray gun. Seedlings wereair-dried for approximately 45 minutes and incubated under a sealed lidat 100% relative humidity in a growth chamber at 16° C. with 9 hours oflight per day (100 mE/m2/s). The sporulation levels were quantified 7days post inoculation (dpi) by counting the number of sporangiophoresper seedling, for at least 40 seedlings per tested line (FIG. 6a ) or byisolating spores in water 5 dpi and determining the spore concentrationto give the number per mg leaf tissue (FIG. 6b ).

Generation of Backcrossed Dmr6 Lines

The dmr6 mutants were back crossed twice (BC2) to the parental line Lereds1-2 as well as Ler. The BC2 lines generated with Ler were selectedfor the presence of the wild type EDS1 gene by PCR analysis.

Cloning DMR6

Fine mapping of the dmr6 gene was done with PCR markers designed usingthe Cereon database to identify insertion and deletion (IND) differencesbetween Col-0 and Ler. The markers: IND_MOP9 in gene At5G24210;IND_K16H17 in gene At5G24420; IND_T4C12 in gene At5G24820; IND_T11H3 inbetween genes At5G24950_60 and IND_F21J6 in gene At5G25270 were used formapping (Table 2). An additional screen for new recombinants wasinitiated on 300 F₂ plants resulting in eight F₂ recombinant plantsbetween the two IND based markers IND_MOP9 and IND_T4C12, which flankeda region of 61 genes. Seven additional markers (M450-M590; Table 2)reduced the region to eighteen candidate genes for the dmr6 locus,between At5g24420 and At5g24590. Sequence analysis of At5g24530indicated a point mutation leading to a stop codon in exon 2 in thedmr6-1 mutant.

Identification of a Dmr6 T-DNA Insertion Line

A second dmr6 allele was identified, 445D09 a FLAG T-DNA insertion linegenerated by INRA Versailles in the Ws-4 accession background. The T-DNAinsertion was confirmed by PCR using a primer designed in the At5g24530gene, LP primer (5′-caggtttatggcatatctcacgtc-3′) (SEQ ID NO: 108), incombination with the T-DNA right border primer, Tag3′(5′-tgataccagacgttgcccgcataa-3′) (SEQ ID NO: 109) or RB4(5′-tcacgggttggggtttctacaggac-3′) (SEQ ID NO: 110). The exact T-DNAinsertion in the second intron of At5g24530 was confirmed by sequencingof amplicons generated with the T-DNA primers from both the left andright border in combination with the gene specific primers LP or RP(5′-atgtccaagtccaatagccacaag-3′) (SEQ ID NO: 111).

cDNA Synthesis

RNA was isolated (from approximately 100 mg leaf tissue from 10 day oldseedlings) with the RNaesy kit (Qiagen, Venlo, The Netherlands) andtreated with the RNase-free DNase set (Qiagen). Total RNA was quantifiedusing an UVmini-1240 spectrophotometer (Shimadzu, Kyoto, Japan). cDNAwas synthesized with Superscript III reverse transcriptase (Invitrogen,Carlsbad, Calif., USA) and oligo(dT)15 (Promega, Madison, Wis., USA),according manufactures instructions.

Complementation of the Dmr6-1 Mutant

Complementation lines were generated by transforming dmr6 plants by thefloral dip method with Agrobacterium tumefaciens (Clough and Bent, 1998)containing the At5g24530 gene from Col-0 behind the 35S promoter. Theconstruct was generated by PCR amplification of the full lengthAt5g24530 from Col-0 cDNA with primers which included restriction sitesthat were used for directional cloning. A forward primer(5′-ttctgggatccaATGGCGGCAAAGCTGATATC-3′) (SEQ ID NO: 1) containing aBamHI restriction site near the start codon (ATG), amplified the 5′-endof DMR6 and at the 3′-end after the stop codon an EcoRI site wasgenerated with a reverse primer(5′-gatatatgaattcttagttgtttagaaaattctcgaggc-3′) (SEQ ID NO: 2). The35S-DMR6-Tn was cloned into the pGreenII0229 (Hellens, R. P., Edwards,E. A., Leyland, N. R., Bean, S., and Mullineaux, P. M. (2000)). pGreen:a versatile and flexible binary Ti vector for Agrobacterium-mediatedplant transformation. Plant Mol. Biol. 42, 819-832). 300 μMDL-Phosphinothricin (BASTA) resistant seedlings were isolated andanalyzed for H. parasitica susceptibility and for DMR6 expression levelsby RT-PCR.

Knock Down Lines of DMR6 by RNAi

RNAi lines were generated in the Ler eds1-2 and Col-0 background. A 782bp long cDNA amplicon of Col-0 At5g24530 gene was generated. The PCR wasdone with the Phusion DNA polymerase (2U/μL) and two different primercombinations. The amplicon from the first DMR6 gene specific primercombination

(RNAiDMR6F: 5′-aaaaagcaggctGACCGTCCACGTCTCTCTGAA-3′ (SEQ ID NO: 3) andRNAiDMR6R: 5′-AGAAAGCTGGGTGAAACGATGCGACCGATAGTC-3′) (SEQ ID NO: 4)was used as a template for the second PCR amplification with generalprimers allowing recombination into the pDONR7 vector of the GateWaycloning system. For the second PCR 10 μl of the first PCR (denaturationfor 30 sec. at 98° C. followed by 10 cycles of: 10 sec. at 98° C.; 30sec. at 58° C.; 30 sec. at 72° C.) in a total volume of 20 μl was usedas template. The second PCR (denaturation for 30 sec. at 98° C. followedby 5 cycles of: 10 sec. at 98° C.; 30 sec. at 45° C.; 30 sec. at 72° C.and 20 cycles of 10 sec. at 98° C.; 30 sec. at 55° C.; 30 sec. at 72° C.finished by a final extension of 10 min. at 72° C.) with the attB1(5′-GGGACAAGTTTGTACAAAAAAGCAGGCT-3′) (SEQ ID NO: 5) and the attB2

(SEQ ID NO: 6) (5′-ggggaccactttgtacaagaaagctgggt-3′)were performed in a 50 μl reaction volume. PCR product was gel purifiedand 50

g insert was recombined into 150

g pDONR7 vector with the clonase BP enzyme. The vector was transformedinto electrocompotent DHSα E. coli cells and plasmids containing thecorrect insert were isolated and 100

g of the pDONR7 with the DMR6 amplicon were used in the LR reaction torecombine the insert in two opposite direction into 150

g pHellsgate8 vector. After transformation into E. coli, Spectomycinresistant clones were selected and the isolated plasmids were verifiedby a NotI digest for the right insert size and by colony PCR with asingle internal primer for At5G24530 (DfragmentF:5′-gagaagtgggatttaaaatagaggaa-3′) (SEQ ID NO: 7), if the inserts wasinserted twice in opposite direction an amplicon of 1420 bp could bedetected. Correct pHellsgate8 plasmids with the double insert inopposite directions were transformed into electrocompotent Agrobacteriumstrain, C58C1. Plasmids were isolated from the Agrobacterium andretransformed into the E. coli to confirm the right size of the plasmidand the insert by NotI digestion. The reconfirmed Agrobacterium strainswere used for the floral dip transformation of the Col-0 and Ler eds1-2plants. The developed seeds were screened for Kanamycin resistance on ½×GM plates, the T₁ seedlings were transferred and the next generation ofseeds the T2 was analysed for DMR6 expression and H. parasiticasusceptibility.

Gene Expression Profiling of the Dmr6 Mutant

Total RNA was isolated as described above. mRNA was amplified with theMessageAmp aRNA kit (Ambion). CATMA array (Crowe et al., 2003) slidescontaining approximately 25,000 gene specific tags were hybridizedaccording to standardized conditions described by de Jong et al. (deJong M., van Breukelen B., Wittink, F. R., Menke, F. L., Weisbeek, P.J., and Van den Ackerveken G. (2006). Membrane-associated transcripts inArabidopsis; their isolation and characterization by DNA microarrayanalysis and bioinformatics. Plant J. 46, 708-721). For quantitativePCR, cDNA templates were generated as described previously. Cyclethresholds were determined per transcript in triplicate using the ABIPRISM 7700 sequence detection system (Applied Biosystems, Foster City,Calif., USA) using SYBR Green I (Applied Biosystems, Foster City,Calif., USA) as reporter dye. Primer sets for the transcripts are DMR6(QDMR6F:5′-TGTCATCAACATAGGTGACCAG-3′ (SEQ ID NO: 8) and QDMR6R:5′-CGATAGTCACGGATTTTCTGTG-3′) (SEQ ID NO: 9), At1g14880(QAt1g14880F:5′-CTCAAGGAGAATGGTCCACA-3′ (SEQ ID NO: 10) and QAt1g14880R:5′-CGACTTGGCCAAATGTGATA-3′) (SEQ ID NO: 11), At4g14365 (QAt4g14365F:5′-TGGTTTTCTGAGGCATGTAAA-3′ (SEQ ID NO: 12) andQAt4g14365R:5′-AGTGCAGGAACATTGGTTGT-3′) (SEQ ID NO: 13), ACD6(QACD6F:5′-TGGACAGTTCTGGAGCAGAT-3′ (SEQ ID NO: 14) and QACD6R:5′-CAACTCCTCCGCTGTGAG-3′) (SEQ ID NO: 15), PR-5(QPR-5F:5′-GGCAAATATCTCCAGTATTCACA-3′ (SEQ ID NO: 16) and QPR-5R:5′-GGTAGGGCAATTGTTCCTTAGA-3′) (SEQ ID NO: 17), PR-2 (QPR-2F:5′-AAGGAGCTTAGCCTCACCAC-3′ (SEQ ID NO: 18) and QPR-2R:5′-GAGGGAAGCAAGAATGGAAC-3′) (SEQ ID NO: 19), PR-1(QPR-1F:5′-GAACACGTGCAATGGAGTTT-3′ (SEQ ID NO: 20) and QPR-1R:5′-GGTTCCACCATTGTTACACCT-3′) (SEQ ID NO: 21) and ΔCT-2 (QACT2F:5′-AATCACAGCACTTGCACCA-3′ (SEQ ID NO: 22) and QACT2R:5′-GAGGGAAGCAAGAATGGAAC-3′) (SEQ ID NO: 23) generating 100 base pairfragments.

Results Characterization of the Gene Responsible for Pathogen Resistancein the Dmr6 Mutant

Van Damme et al., 2005, supra disclose a dmr6 mutant that is resistantto H. parasitica. The level of resistance can be examined by countingthe number of sporangiophores per seedling seven day post inoculationwith the H. parasitica (isolate Waco9 or Cala2, obtainable from Dr. G.Van den Ackerveken, Plant-Microbe Interactions Group, University ofUtrecht, Utrecht, NL). The parental line, Ler eds1-2 (Parker et al.,1996, Plant Cell 8:2033-2046), which is highly susceptible, is used as apositive control (and is set at 100%).

The reduction in sporangiophore formation on the infected dmr6 mutantscompared to seedlings of the parental lines is shown in FIG. 6a ,wherein the results of the quantification of Hyaloperonosporaparasitica, Waco9 sporulation (sporangiophores/seedling) on the downymildew resistant dmr6-1 mutant, back-crossed twice to the parental lineLer eds1-2, and on mutant dmr6-2 (FLAG 445D09 T-DNA line) compared tothe control lines is shown.

According to the invention, the gene responsible for resistance to H.parasitica in the dmr6 mutants of van Damme et al., 2005, supra, hasbeen cloned by a combination of mapping and sequencing of candidategenes. Previously, the recessive dmr6 mutation was mapped near thenga139 marker on chromosome 5 to a region encompassing 74 genes. Finemapping linked the dmr6 locus to a mapping interval containing the BACsT13K7 and K18P6 between the markers At5g24420 and At5g24590 located inthe corresponding genes. This allowed the dmr6 interval to be confinedto a region of 18 candidate genes. Comparative sequence analysis of the18 genes in dmr6 and the parental line, Ler eds1-2 revealed a pointmutation in the second exon of the At5g24530 gene. This single basechange of G to A, typical for an EMS mutation, changes a TGG a (trpcodon) to a TGA (premature stop codon) at nucleotide position 691 of thecoding sequence (FIG. 7). The early stop codon truncates the predictedoxidoreductase enzyme of 342 aa at position 141 before the conservedcatalytic domain suggesting that dmr6 is a null-allele. The At5g24530coding sequence (FIG. 2) is predicted to encode a protein with a mass of39.4 kDa. No biological role has so far been described for At5g24530.

At5g24530 is DMR6

A second allele, dmr6-2, was identified in a T-DNA insertion line(FLAG_445D09) from the mutant collection from INRA, Versailles. Thepresence and location of the T-DNA insert in the second intron ofAt5g24530 (FIG. 7) was confirmed by PCR and sequence analysis (data notshown). Progeny of the FLAG 445D09 line homozygous for the T-DNAinsertion was resistant to H. parasitica isolate Waco9, whereas theparental line (Ws-4) was susceptible (FIG. 6a ). The At5g24530transcript could be amplified by RT-PCR using primers in exon 2 and 3 inWs-4, but not in the homozygous dmr6-2 line (data not shown), indicatingthat dmr6-2 can be considered a second null-allele.

To corroborate the idea that At5g24530 is required for susceptibility toH. parasitica the dmr6-1 mutant was transformed with the cDNA fromAt5g24530 cloned under control of the 35S promoter. In five independentdmr6-1 T2 seedlings the strong overexpression of At5g24530 was confirmedby RT-PCR (data not shown). All T3 lines, homozygous for the transgene,showed restoration of susceptibility to H. parasitica isolate Cala2(FIG. 6b ), confirming that At5g24530 is DMR6. The complementation,together with the identification of two independent dmr6 mutants clearlyindicates that a functional DMR6 gene is required for susceptibility toH. parasitica.

DMR6 is Transcriptionally Activated During H. Parasitica Infection

To study the expression of DMR6 during infection with H. parasiticarelative transcript levels were measured by quantitative PCR at sixdifferent time points from 0 days (2 hours) post inoculation to 5 dayspost inoculation (dpi) (FIG. 8). RNA was isolated from ten day old Lerseedlings that were spray inoculated with water (mock), compatible, orincompatible H. parasitica isolate. At 2 hours post inoculation (0 dpi)the levels of DMR6 transcripts were equal in the different treatments.Starting from 1 dpi, the level of DMR6 transcript was significantlyincreased in both the compatible and incompatible interaction comparedto mock-treated seedlings. The DMR6 transcript level was slightly butsignificantly higher at 1 dpi in the incompatible interaction (ΔCT of3.5, approximately 11 fold induction) than in the compatible (ΔCT of3.0, approximately 8 fold induction). The expression level increasedfurther in time to reach a stable high level at 4-5 dpi. At these timepoints the DMR6 transcript level was higher in the compatible than inthe incompatible interaction. The elevated DMR6 transcript levels duringcompatible and incompatible H. parasitica interactions suggest a role ofDMR6 in plant defence. The defence-associated expression of DMR6 couldbe confirmed in our three enhanced-defence mutants, dmr3, dmr4, and dmr5(Van den Ackerveken et al., unpublished). Furthermore, in silicoanalysis of DMR6 levels in the Genevestigator Mutant Surveyor(Zimmermann, P., Hennig, L., and Gruissem, W. (2005). Gene-expressionanalysis and network discovery using Genevestigator. Trends Plant Sci.10, 407-409) showed that the gene is strongly induced in the pathogenresistant mutants mpk4 and cpr5. In the cpr5/npr1 double mutant the DMR6transcript level remained high indicating that the induction of DMR6expression is mostly NPR1 independent. Salicylic acid appears to be animportant signal in the induction of DMR6 expression during senescenceas nahG transgenic plants (expressing the bacterial salicylatehydroxylase gene) showed only low levels of DMR6 transcript.

To investigate in more detail how the expression of DMR6 is activatedduring biotic and abiotic stress, DMR6 reporter lines were generated.The localisation of DMR6 expression was studied in transgenic Col-0 andLer eds1-2 plants containing the DMR6 promoter linked to the uidA(β-glucuronidase, GUS) reporter gene (pDMR6::GUS). To visualise both H.parasitica hyphal growth, by staining with trypan blue, as well as GUSactivity, magenta-Xgluc was used as a β-glucuronidase substrate yieldinga magenta precipitate. In uninfected plants no GUS expression could bedetected in the different plant organelles; roots, meristem, flower,pollen and seed. The expression of DMR6 was induced in the compatibleinteractions, Ler eds1-2 infected with Cala2 (FIG. 9a ), and Col-0infected with isolate Waco9 (FIG. 9b ). GUS expression was also inducedin the incompatible interaction Ler eds1-2 inoculated with isolate Emoy2(FIG. 9c ). As shown in FIGS. 9a and 9b DMR6 expression was confined tothe cells in which H. parasitica had formed haustoria. Plant cellscontaining the most recently formed haustoria did not show detectablelevels of GUS activity (FIG. 9a , indicated by asterisk). During theincompatible interaction (FIG. 9c ) activity of the DMR6 promoter couldonly be detected in the cells that were in contact with the initialinvading hyphae. In death cells, resulting from the hypersensitiveresponse (HR, visualized by trypan blue staining indicated in FIG. 9c byasterisk) no GUS activity could be detected, possibly due to proteindegradation in these cells. To test if the DMR6 expression inhaustoria-containing cells is caused by a wound-like response, seedlingswere wound by incision with scissors and stained for GUS activity 3 dayslater. No detectable promoter DMR6 GUS expression was seen, indicatingthat the expression of DMR6 is not induced by wounding (FIG. 9d ).Furthermore the local induction of DMR6 expression was tested inresponse to treatment with benzothiadiazole (BTH), a functional analogueof salicylic acid (SA). At 3 days post BTH treatment GUS activity wasmainly localized in the newly formed, but not in the mature leaves (FIG.9e ). Analysis of pDMR6::GUS lines confirm the expression data describedabove and highlights the strictly localized induction of DMR6 inresponse to H. parasitica infection.

The Dmr6-1 Mutant Constitutively Expresses Defence AssociatedTranscripts

To elucidate how the lack of DMR6 results in H. parasitica resistance,the transcriptome of the dmr6-1 mutant compared to the Ler eds1-2parental line was analysed. Probes derived from mRNA of the above-groundparts of 14 day old dmr6-1 and Ler eds1-2 seedlings were hybridised onwhole genome CATMA micro arrays. A total of 58 genes were found to besignificantly differentially expressed in dmr6-1, of which 51 genes hadelevated and 7 genes had reduced transcript levels. A pronounced set ofthe 51 induced transcripts have been identified as genes associated withactivated plant defence responses, e.g., ACD6, PR-5, PR-4/HEL and PAD4.These data indicate that the loss of DMR6 results in the activation of aspecific set of defence-associated transcripts. The finding that DMR6 isamong the dmr6-1-induced genes corroborates the idea that DMR6 isdefence-associated. To test if the induced expression of thedefence-associated genes was due to the loss of DMR6 and not due toadditional ethane methyl sulfonate (EMS) mutations remaining in thebackcrossed dmr6-1 mutant the transcript level of a selection of genes(At4g14365, At1g14880, ACD6, PR-1, PR-2 and PR-5) was verified byquantitative PCR in both the dmr6-1 and dmr6-2 mutant (FIG. 10). Wecould only test DMR6 transcript levels in the dmr6-1 mutant (FIG. 10a )as the dmr6-2 mutant (FIG. 10b ) has a T DNA insertion disrupting theDMR6 transcript. The induction of DMR6 as observed in the micro arrayanalysis was confirmed by Q-PCR in dmr6-1 compared to Ler eds1-2 (FIG.10a ). FIGS. 10a and b show that all six selected genes were elevated inboth dmr6 mutants compared to the parental lines. The observed elevatedexpression of the selected defence-associated genes in the dmr6 mutantsindicates that lack of DMR6 activates a plant defence response. Theactivation of this set of defence-associated transcripts could be theprimary cause of resistance to H. parasitica in the dmr6 mutants.

Example 2

Identification of DMR6 orthologs in crops 1. Screening of libraries onthe basis of sequence homology

The nucleotide and amino acid sequences of the DMR6 coding sequence andprotein of Arabidopsis thaliana are shown in FIG. 2. Public libraries ofnucleotide and amino acid sequences were compared with the sequences ofFIG. 2. This comparison resulted in identification of the complete DMR6coding sequences and predicted amino acid sequences in Aquilegiaspecies, Citrus sinensis, Coffea canephora, Cucumis sativus, Gossypiumhirsitum, Lactuca sativa, Medicago truncatula, Oryza sativa (3), Populustrichocarpa (2), Solanum lycopersicum (2), Sorghum bicolor, Spinaciaoleracea, Vitis vinifera, Zea mays, and Zingiber officinale. Thesequence information of the orthologous proteins thus identified isgiven in Table 1 and visualized in an multiple alignment in FIG. 1. Formany other plant species orthologous DNA fragments could be identifiedby BlastX as reciprocal best hits to the Arabidopsis or other plant DMR6protein sequences.

2. Identification of Orthologs by Means of Heterologous Hybridisation

The DMR6 DNA sequence of Arabidopsis thaliana as shown in FIG. 2 is usedas a probe to search for homologous sequences by hybridization to DNA ofany plant species using standard molecular biological methods. Usingthis method orthologous genes are detected by southern hybridization onrestriction enzyme-digested DNA or by hybridization to genomic or cDNAlibraries. These techniques are well known to the person skilled in theart. As an alternative probe the DMR6 DNA sequence of any other moreclosely related plant species can be used as a probe.

3. Identification of Orthologs by Means of PCR

For many crop species, partial DMR6 mRNA or gene sequences are availablethat are used to design primers to subsequently PCR amplify the completecDNA or genomic sequence. When 5′ and 3′ sequences are available themissing internal sequence is PCR amplified by a DMR6 specific 5′ forwardprimer and 3′ reverse primer. In cases where only 5′, internal or 3′sequences are available, both forward and reverse primers are designed.In combination with available plasmid polylinker primers, inserts areamplified from genomic and cDNA libraries of the plant species ofinterest. In a similar way, missing 5′ or 3′ sequences are amplified byadvanced PCR techniques; 5′RACE, 3′ RACE, TAIL-PCR, RLM-RACE orvectorette PCR.

As an example the sequencing of the Lactuca sativa (lettuce) DMR6 cDNAis provided. From the Genbank EST database at NCBI several Lactuca DMR6ESTs were identified using the tblastn tool starting with theArabidopsis DMR6 amino acid sequence. Clustering and alignment of theESTs resulted in a consensus sequence for a 5′ DMR6 fragment. To obtainthe complete lettuce DMR6 cDNA the RLM-RACE kit (Ambion) was used onmRNA from lettuce seedlings. The 3′ mRNA sequence was obtained by usingtwo primers that were designed in the 5′ DMR6 consensus sequence derivedfrom ESTs (Lsat_dmr6_fw1: CGATCAAGGTCAACACATGG (SEQ ID NO: 24), andLsat_dmr6_fw2: TCAACCATTACCCAGTGTGC) (SEQ ID NO: 25) and the 3′RACEprimers from the kit. Based on the assembled sequence new primers weredesigned to amplify the complete DMR6 coding sequence from cDNA toprovide the nucleotide sequence and derived protein sequence aspresented in FIG. 3.

The complete DMR6 coding sequences from more than 10 different plantsspecies have been identified from genomic and EST databases. From thealignment of the DNA sequences, conserved regions in the coding sequencewere selected for the design of degenerate oligonucleotide primers (forthe degenerate nucleotides the abbreviations are according to the IUBnucleotide symbols that are standard codes used by all companiessynthesizing oligonucleotides; G=Guanine, A=Adenine, T=Thymine,C=Cytosine, R=A or G, Y=C or T, M=A or C, K=G or T, S=C or G, W=A or T,B=C or G or T, D=G or A or T, H=A or C or T, V=A or C or G, N=A or C orG or T).

The procedure for obtaining internal DMR6 cDNA sequences of a givenplant species is as follows:

1. mRNA is isolated using standard methods,2. cDNA is synthesized using an oligo dT primer and standard methods,3. using degenerate forward and reverse oligonucleotides a PCR reactionis carried out,4. PCR fragments are separated by standard agarose gel electrophoresisand fragments of the expected size are isolated from the gel,5. isolated PCR fragments are cloned in a plasmid vector using standardmethods,6. plasmids with correct insert sizes, as determined by PCR, areanalyzed by DNA sequencing,7. Sequence analysis using blastX reveals which fragments contain thecorrect internal DMR6 sequences,8. The internal DNA sequence can then be used to design gene- andspecies-specific primers for 5′ and 3′ RACE to obtain the complete DMR6coding sequence by RLM-RACE (as described above).

As an example the sequencing of the Cucumis sativus (cucumber) DMR6 cDNAis provided. For cucumber several primer combinations between thefollowing primers were successful in amplifying a stretch of internalcoding sequence from cDNA; forward primers dmr6_deg_fw1B(TTCCAGGTDATTAAYCAYGG) (SEQ ID NO: 26), dmr6_deg_fw2BCATAAYTGGAGRGAYTAYCT) (SEQ ID NO: 27), dmr6_deg_fw3B(GARCAAGGRCARCAYATGGC) (SEQ ID NO: 28) and dmr6_deg_fw4(AATCCTCCTTCHTTCAAGGA) (SEQ ID NO: 29) and reverse primers dmr6_deg_rv3B(AGTGCATTKGGGTCHGTRTG) (SEQ ID NO: 30), dmr6_deg_rv4(AATGTTRATGACAAARGCAT) (SEQ ID NO: 31) and dmr6_deg_rv5(GCCATRTGYTGYCCTTGYTC) (SEQ ID NO: 32). After cloning and sequencing ofthe amplified fragments cucumber DMR6-specific primers were designed for5′ RACE (Cuc_dmr6_rv1: TCCGGACATTGAAACTTGTG (SEQ ID NO: 33) andCuc_dmr6_rv2: TCAAAGAACTGCTTGCCAAC) (SEQ ID NO: 34) and 3′ RACE(Cuc_dmr6_fw1: CGCACTCACCATTCTCCTTC (SEQ ID NO: 35) and Cuc_dmr6_fw2:GGCCTCCAAGTCCTCAAAG) (SEQ ID NO: 36). Finally the complete cucumber DMR6cDNA sequence was amplified and sequenced (FIG. 5). A similar approachwas a used for spinach, Spinacia oleracea (FIG. 4), Solanum lycopersicum(FIG. 12) and Nicotiana benthamiana (FIG. 13).

Orthologs identified as described in this example can be modified usingwell-known techniques to induce mutations that reduce the DMR6expression or activity, to obtain non-genetically modified plantsresistant to Fungi or Oomycota. Alternatively, the genetic informationof the orthologs can be used to design vehicles for gene silencing, andto transform the corresponding crop plants to obtain plants that areresistant to Oomycota.

Example 3 Mutation of Seeds

Seeds of the plant species of interest are treated with a mutagen inorder to introduce random point mutations in the genome. Mutated plantsare grown to produce seeds and the next generation is screened for theabsence of reduction of DMR6 transcript levels or activity. This isachieved by monitoring the level of DMR6 gene expression, or bysearching for nucleotide changes (mutations) by the TILLING method, byDNA sequencing, or by any other method to identify nucleotide changes.The selected plants are homozygous or are made homozygous by selfing orinter-crossing. The selected homozygous plants with absent or reducedDMR6 transcript activity are tested for increased resistance to thepathogen of interest to confirm the increased disease resistance.

Example 4

Transfer of a Mutated Allele into the Background of a Desired Crop

Introgression of the desired mutant allele into a crop is achieved bycrossing and genotypic screening of the mutant allele. This is astandard procedure in current-day marker assistant breeding of crops.

Example 5 Use of the DMR6 Promotor for Pathogen-Induced Gene Expressionand the Generation of Disease Resistant Plants

Precise control of transgene expression is pivotal to the engineering ofplants with increased disease resistance. In the past, constitutiveoverexpression of transgenes frequently has resulted in poor qualityplants. It has therefor been suggested to use pathogen-induciblepromotors, by which the transgenes are expressed only when and wherethey are needed—at infection sites.

Local and inducible expression of engineered genes, e.g. master switchgenes, elicitor or Avr genes, anti-microbial genes, or toxic genes,results in the activation of defence or cell death that will lead topathogen resistance, such as described by Gurr and Rushton (Trends inBiotechnology 23: 275-282, 2005). A good example is provided by De wit(Annu. Rev. Phytopathol. 30: 391-418, 1992) who proposes the use of theAvr9-Cf9 combination to achieve induced cell death leading to diseaseresistance. The tissue-specificity and inducibility of expression is ofprime importance for such approaches, as described by Gun and Rushton(Trends in Biotechnology 23: 283-290, 2005).

According to the present invention, the DMR6 promoter has beendemonstrated to show a strong, inducible, localized expression based onpromoter-GUS analysis. Thus, the DMR6 promotor is very suitable forengineering disease resistance in transgenic plants. The DMR6 promoterconsists of a region of 2.5 kb that is upstream of the Arabidopsis DMR6coding sequence (ATG start codon) and includes the 5′UTR (as depicted inFIG. 11). This pathogen-inducible promotor is then used to engineersuitable transgene constructs, using standard techniqus known the personskilled in the art.

Using orthologous DNA sequences from a given plant species primers aredesigned for PCR. These are then used to screen genomic libraries of theplant species of interest to identify the genomic clones that containthe DMR6 ortholog with its promoter and regulatory sequences.Alternatively, the genomic clones are isolated by screening a librarywith a labelled PCR fragment corresponding to the DMR6 orthologous gene.Sequencing reveals the nucleotide sequence of the promoter. The regionof 2-5 kb upstream the DMR6 orthologous coding sequence (ATG startcodon), so including the 5′UTR, is then amplified by PCR to engineertransgene constructs for plant transformation.

Example 6

This example demonstrates the complementation of mutant dmr6-1 inArabidopsis thaliana by DMR6 orthologs from 4 different crop species.For this, DMR6 orthologs of Cucumis sativa (Cs), Spinacia oleracea (So),Lactuca sativa (Ls) and Solanum lycopersicum (Sl) were cloned into aplant expression vector under the control of the 35S promoter and,subsequently, this vector was transformed into a Arabidopsis thalianamutant dmr6-1.

Briefly, mRNA was isolated using standard methods and cDNA wassynthesized using an oligo dT primer and standard methods. Subsequently,PCR fragments were generated using primer pairs for each crop asdepicted in table 3 below. The generated PCR products were cloned into apENTR/D-TOPO vector using the pENTR/D-TOPO cloning kit from Invitrogenand resulting plasmids with correct insert sizes, as determined by PCR,were analyzed by DNA sequencing. Recombination to the pB7WG2,0 vectorwas done using LR clonase II from Invitrogen and the resulting plasmidswere analyzed by PCR and digestion with restriction enzymes. Suitableplasmids were transformed into Agrobacterium tumefaciens C58C1 PGV2260and plasmids from Agrobacterium were analyzed by PCR and digestion withrestriction enzymes.

Arabidopsis thaliana dmr6-1 plants were transformed with the aboveconstructs by dipping into Agrobacterium solution and overexpression ofcrops DMR6 in Arabidopsis T1 plants is verified by RT-PCR using thecrops DMR6 cloning primers (table 3). Finally, Arabidopsis T2 and T3plants were infected with Hyaloperonospora parasitica Cala2 to confirmcomplementation. The results are shown in FIG. 14.

As shown in FIG. 14, all DMR6 orthologs tested were capable ofcomplementing Arabidopsis thaliana mutant dmr6-1 indicating that theDMR6 orthologs identified encode DMR6 proteins with a similarfunctionality as Arabidopsis thaliana DMR6.

Tables

Table 1 lists the GI numbers (GenInfo identifier) and Genbank accessionnumber for Expressed Sequence Tags (ESTs) and mRNA or protein sequencesof the Arabidopsis DMR6 mRNA and orthologous sequences from other plantspecies. A GI number (genInfo identifier, sometimes written in lowercase, “gi”) is a unique integer which identifies a particular sequence.The GI number is a series of digits that are assigned consecutively toeach sequence record processed by NCBI. The GI number will thus changeevery time the sequence changes. The NCBI assigns GI numbers to allsequences processed into Entrez, including nucleotide sequences fromDDBJ/EMBL/GenBank, protein sequences from SWISS-PROT, PIR and manyothers. The GI number thus provides a unique sequence identifier whichis independent of the database source that specifies an exact sequence.If a sequence in GenBank is modified, even by a single base pair, a newGI number is assigned to the updated sequence. The accession numberstays the same. The GI number is always stable and retrievable. Thus,the reference to GI numbers in the table provides a clear andunambiguous identification of the corresponding sequence.

TABLE 1 Species Common name Detail GI number Genbank Arabidopsisthaliana Thale cress mRNA 42568064 NM_122361 Aquilegia_sp Aquilegia ESTs75461114 DT768847.1 74538666 DT745001.1 74562677 DT760187.1 75461112DT768846.1 74562675 DT760186.1 Citrus sinensis Sweet Orange ESTs 5793134CX672037.1 57933368 CX673829.1 63078039 CX309185.1 Coffea canephoraCoffea ESTs 82485203 DV705375.1 82458236 DV684837.1 82461999 DV688600.182487627 DV707799.1 Gossypium hirsutum Cotton ESTs 109842586 DW241146.148751103 CO081622.1 Sorghum bicolor Sorghum ESTs 45992638 CN150358.157813436 CX614669.1 45985339 CN145819.1 57821006 CX622219.1 45989371CN148311.1 57821495 CX622708.1 45959033 CN130459.1 45985193 CN145752.118058986 BM322209.1 45958822 CN130381.1 30164583 CB928312.1 Medicagotruncatula Barrel medic Genome MtrDRAFT_AC119415g1v1 draft protein92878635 ABE85154 Oryza sativa 1 Rice Genome OSJNBb0060I05.4 protein18057095 AAL58118.1 Oryza sativa 2 mRNA 115450396 NM_001055334 protein115450397 NP_001048799 Oryza sativa 3 mRNA 115460101 NM_001060186protein 115460102 NP_001053651 Populus trichocarpa 1 Poplar Genome:LG_XII: 3095392-3103694 protein: Poptr1_1: 569679, eugene3.00120332Populus trichocarpa 2 Poplar Genome: LG_XV: 201426-209590 protein:Poptr1_1: 732726, estExt_Genewise1_v1.C_LG_XV0083 Solarium lycopersicum1 Tomato ESTs 62932307 BW689896.1 58229384 BP885913.1 117682646DB678879.1 5894550 AW035794.1 117708809 DB703617.1 62934028 BW691617.115197716 BI422913.1 4381742 AI486371.1 5601946 AI896044.1 4387964AI484040.1 4383017 AI487646. 5278230 AI780189.1 12633558 BG133370.176572794 DV105461.1 117692514 DB718569.1 4385331 AI489960.1 4383253AI487882.1 4384827 AI489456.1 Solarium lycopersicum 2 Tomato ESTs47104686 BT013271.1 14685038 BI207314.1 14684816 BI207092.1 Zea maysMaize ESTs 110215403 EC897301.1 76291496 DV031064.1 91050479 EB160897.191874282 EB404239.1 110540753 EE044673.1 78111856 DV530253.1 94477588EB706546.1 71441483 DR822533.1 78111699 DV530096.1 78107139 DV525557.176017449 DT944619.1 91048249 EB158667.1 78104908 DV523326.1 78088214DV516607.1 76291495 DV031063.1 71441482 DR822532.1 78088213 DV516606.1Vitis vinifera Grape ESTs 33396402 CF202029.1 33399765 CF205392.145770972 CN006824.1 45770784 CN006636.1 45770528 CN006380.1 45770631CN006483.1 33400623 CF206250.1 33396335 CF201962.1 30134763 CB920101.130305300 CB982094.1 71857419 DT006474.1 30305235 CB982029.1 Zingiberofficinale Ginger ESTs 87108948 DY375732.1 87095447 DY362231.1 87095448DY362232.1 87094804 DY361588.1 87095449 DY362233.1 87094803 DY361587.1Lactuca sativa Lettuce Sequence described in this patent applicationSpinacia oleracea Spinach Sequence described in this patent applicationCucumis sativus Cucumber Sequence described in this patent applicationNicotiana benthamiana Tobacco Sequence described in this patentapplication

TABLE 2Primer sequences of insertion/deletion markers (size difference in brackets)used in the mapping and cloning of the DMR6 gene. INDEL/ Name primerGene enzyme Forward primer Reverse primer IND_MOP9 At5G24210tttgggaacagaaaaagt catattcaaaagggaaaatc tggaggt (SEQ IDccaga (SEQ ID NO: NO: 37) 38) IND_K16H17 At5g24420 tggggttgtggtttattctgtggccaatagtagttgatac ttgac (SEQ ID gcaaga (SEQ ID NO: 39) NO: 40)IND_T4C12 At5g24820 tctcgggtaagacacaa tattccaacttgcgacgtaggtcgagat (SEQ ID agcat (SEQ ID NO: NO: 41) 42) IND_T11H3 At5g24950-60ccaattgggttatttacttc cggcttttaacaacatattttc gat (SEQ ID NO:ca (SEQ ID NO: 44) 43) IND_F21J6 At5g25270 aacacatcaccaagatgcctctgccccaagaaatatt aatccaga (SEQ ID gagat (SEQ ID NO: NO: 45) 46) M450At5G24450 18 agctttgtatggtagtgcc gcggtatacgggggttaaa aatga (SEQ IDatcta (SEQ ID NO: NO: 47) 48) M490 At5g24490 TaqI atggccaaccactctttgtacaagcaagaagaacagc tac (SEQ ID NO: gaag (SEQ ID NO: 49) 50) M525At5g24520-30 TaqI gaaatttggttgttggcat tcaagatcttcatattctcattttatc (SEQ ID NO: cca (SEQ ID NO: 52 51) M545 At5G24540/50 41cagctgaagtatgtttcat cttgcaattgttgggactag cccttt (SEQ ID gtaa (SEQ ID NO:NO: 53) 54) M555 At5G24550/60 14 tcactaaccagtgaaaaa tatacagcgaatagcaaagggttgc (SEQ ID ccaag (SEQ ID NO: NO: 55) 56) M470 At5g24470 HphIccgcgagtgtaatatatct cagtttaacgcatgaagtgc ctcct (SEQ ID NO:tagt (SEQ ID NO: 57) 58) M590 At5g24590 PdmI gcatcatttgtaccgtacttagtggatactctgtccctg gagtc (SEQ ID aggt (SEQ ID NO: NO: 59 60)

TABLE 3 Primer pairs for cloning dmr6 orthologs in a suitable plantexpression vector Arabidopsis thaliana AtDMR6_fw CACCATGGCGGCAAAGCTGATA (SEQ ID NO: 85) AtDMR6UTR_rv GACAAACACAAAGGCCAAAGA (SEQ ID NO: 86)Cucumis sativa cuc_fw CACCATGAGCAGTGTGATGGA GAT (SEQ ID NO: 87)cucUTR_rv TGGGCCAAAAAGTTTATCCA (SEQ ID NO: 88) Spinacia oleracea spi_fwCACCATGGCAAACAAGATATT ATCCAC (SEQ ID NO: 89) spiUTR_rvTTGCTGCCTACAAAAGTACAA A (SEQ ID NO: 90) Lactuca sativa Lsat_fwCACCATGGCCGCAAAAGTCAT CTC (SEQ ID NO: 91) LsatUTR_rvCATGGAAACACATATTCCTTCA (SEQ ID NO: 92) Solanum lycopersicum Slycldmr6_fwCACCATGGAAACCAAAGTTAT TTCTAGC (SEQ ID NO: 93) Slycldmr6UTR_rvGGGACATCCCTATGAACCAA (SEQ ID NO: 94)

1-11. (canceled)
 12. An isolated maize plant that is resistant toPeronosclerospora spp., wherein the maize plant has a reduced level orreduced activity of DMR6 protein as compared to a maize plant that isnot resistant to Peronosclerospora spp., wherein said maize plant has anon-natural mutation introduced into the dmr6 gene encoding the DMR6protein of SEQ ID NO: 83 and said plant has a reduced level or reducedactivity of the DMR6 protein of SEQ ID NO: 83 as compared to the maizeplant that is not resistant to Peronosclerospora spp.
 13. The maizeplant of claim 12, wherein the mutation in the dmr6 gene leads to anamino acid substitution in the DMR6 protein.
 14. The maize plant ofclaim 12, wherein the mutation in the dmr6 gene reduces expression ofthe dmr6 gene and leads to a reduction of endogenous level of the DMR6protein.
 15. A seed, tissue, or plant part of the maize plant of claim12, wherein the seed, tissue, or plant part comprises the mutation inthe dmr6 gene and said plant has a reduced level or reduced activity ofthe DMR6 protein of SEQ ID NO:
 83. 16. A method for obtaining a maizeplant which is resistant to Peronosclerospora spp., the methodcomprising reducing an endogenous level of the DMR6 protein of SEQ IDNO: 83 in a maize plant by introducing a non-natural mutation into thedmr6 gene encoding the DMR6 protein of SEQ ID NO: 83 to produce a maizeplant that is resistant to Peronosclerospora spp.
 17. The method ofclaim 16, wherein reducing the endogenous level of the DMR6 protein inthe maize plant is achieved by reducing expression of the dmr6 gene. 18.The method of claim 17, wherein reducing expression of the dmr6 gene isachieved by gene silencing or RNAi.
 19. The method of claim 16, whereinthe mutation results in one or more amino acid changes that leads to areduced enzymatic activity of the DMR6 protein of SEQ ID NO:
 83. 20. Themethod of claim 16, wherein the mutation is effected by a mutagenictreatment of the maize plant.
 21. The method of claim 20, wherein themutagenic treatment is effected with a mutagen or with radiation.
 22. Amaize plant produced from the method of claim 16, wherein the plantcomprises the mutation in the dmr6 gene and said maize plant has areduced level or reduced activity of the DMR6 protein of SEQ ID NO: 83.23. A seed, tissue, or plant part of the maize plant of claim 22,wherein the seed, tissue, or plant part comprises a mutation in the dmr6gene and has a reduced level or reduced activity of the DMR6 protein ofSEQ ID NO: 83.